Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Apoptosis, the physiologic and genetically modulated process of cell death, is of central importance for modeling tissues and maintaining homeostasis in multicellular organisms (Kerr et al., Br. J. Cancer 26: 239-257, 1972; Jacobson et al., Cell 88: 347-354, 1997). Great progress is being made towards understanding the biochemistry underlying this intrinsic suicide program. The cellular apoptotic effector molecules include a set of cysteine proteinases, termed caspases, that degrade critical cellular substrates (Nicholson et al., Trends Biochem. Sci. 22: 299-306, 1997). The regulatory machinery that governs the activation of the caspases is less well understood. However, a family of proteins of which Bcl-2 is the prototypic molecule (and is referred to as the Bcl-2 family of proteins) plays a central role (Jacobson, Curr. Biol. 7: R277-R281, 1997; Reed, Nature 387: 773-776, 1997; Kroemer, Nature Med. 3: 614-620, 1997; Adams and Cory, Science 281: 1322-1326, 1998).
Bcl-2 was the first intracellular regulator of apoptosis to be identified (Vaux et al., Nature 335: 440-442, 1988) and high levels enhance cell survival under diverse cytotoxic conditions. Other cellular homologs, such as BCl-xL (Boise et al., Cell 74: 597-608, 1993) and Bcl-w (Gibson et al., Oncogene 13: 665675, 1996), also enhance cell survival, as do more distantly related viral homologs, such as the adenovirus E1B 19K protein (White et al., Mol. Cell. Biol. 12: 2570-2580, 1992) and Epstein-Barr virus BHRF-1 (Henderson et al., Proc Natl. Acad. Sci. USA 90: 8479-8483, 1993).
Pro-apoptotic BH3-only members of the Bcl-2 family are essential for initiation of apoptosis in species as distantly related as mice and C. elegans (Huang and Strasser, Cell 103: 839, 2000). EGL-1, the so far only recognized BH3-only protein in C. elegans, is required for all developmentally programmed cell deaths in this organism. In contrast, a number of BH3-only proteins have already been identified in mammals: Blk, Bad, Bik, Hrk, Bid, Bim, Noxa and Puma. Experiments with knock-out mice have shown that different apoptotic stimuli require distinct BH3-only proteins for their initiation. (Huang and Strasser, 2000, supra). For example, Bim is essential for apoptosis induced by cytokine withdrawal or antigen receptor stimulation, but is dispensable for cell death induced by glucocorticoids (Bouillet et al., Science 286: 1735, 1999; Bouillet et al., Nature 415, 922, 2002). In contrast, Bid is involved in Fas-induced killing of hepatocytes (Yin et al., Nature 400: 886, 1999). Moreover, different cell types may require distinct BH3-only proteins for their developmentally programmed death. Consistent with this idea, Bim-deficient mice have an abnormal accumulation of lymphoid and myeloid cells but erythropoiesis appears normal (Bouillet et al., 1999, supra). These results indicate that individual mammalian BH3-only proteins have specific functions.
The pro-apoptotic activity of BH3-only proteins is subject to stringent control. In C. elegans, EGL-1 is regulated by the transcriptional represser TRA-1A in a group of neurons that is required for egg-laying (Conradt and Horvitz, Cell 93: 519, 1998). Some mammalian BH3-only proteins are also subject to transcriptional regulation. For example, Noxa was discovered as a p53-inducible gene and is therefore a prime candidate for mediating DNA damage-induced apoptosis (Oda et al., Science 288: 1053, 2000). Several mammalian BH3-only proteins can also be regulated post-translationally (Huang and Strasser, 2000, supra). In growth factor-stimulated cells, Bad is phosphorylated and sequestered away from pro-survival Bcl-2 family members by binding to 14-3-3 scaffold proteins (Zha et al, Cell 87: 619, 1996). In healthy cells, Bim is sequestered to the microtubular dynein motor complex by binding to dynein light chain, DLC1/LC8 (Puthalakath et al., Mol. Cell 3: 287, 1999). Certain apoptotic stimuli, such as UV-radiation or treatment with taxol, free Bim (still bound to DLC1) and allow it to translocate to, bind and inactivate pro-survival Bcl-2 family members. This process occurs independently of the cell death executioner cysteine proteases (caspases) and therefore constitutes an upstream signalling event in apoptosis (Puthalakath et al., 1999, supra). In contrast, the pro-apoptotic activity of Bid is unleashed upon cleavage by a variety of caspases (e.g. caspase-8) or by the serine protease granzyme B (Li et al., Cell 94: 491-501, 1998; Luo et al., Cell 94: 481-490, 1998), indicating that it functions as part of an amplification mechanism rather than as an initiator of apoptosis. These observations demonstrate that through sequestration to specific sites in the cell, different BH3-only proteins function as sensors for distinct forms of intracellular stress.
In work leading to the present invention, the inventors sought novel BH3-only proteins which played a role in embryogenesis. In accordance with the present invention, the inventors cloned “Bmf” (Bcl-2 modifying factor) which was identified through yeast 2-hybrid screening of a day 17 mouse embryonic library using Mcl-1 as bait. Bmf is proposed to induce cell death and act as a “death-ligand” for certain or all members of the pro-survival Bcl-2 family. The identification of this new gene permits the identification and rational design of a range of products for use in therapy, diagnosis, antibody generation and involving modulation of physiological cell death. These therapeutic molecules may act as either, antagonists or agonists of Bmf's friction and will be useful in cancer, autoimmune or degenerative disease therapy.